Review Reports - Islet Transplantation: Current Limitations and Challenges for Successful Outcomes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Although islet transplantation is a promising treatment option, this review describes the current limitations and options for improvements. Several reviews are available about this topic, Langlois et al. try to be novel in describing new avenues like ER and mitochondrial stress. The review is sometimes difficult to follow and is occasionally missing relevant depth on topics.

1. Is there a word missing at the end of the title? Or should it be ‘challenges for success’? The same for the subtitle: the dark side of islet isolation successful.

2. Authors describe in the first part how ECM is important and that it takes time to restore it post-transplantation, they also describe ischemia during isolation, but nothing is said about the disruption of the blood vessel network and that this also takes time to be restored post-transplantation. Vascularization is mentioned in figure 1, so you also expect it in the text associated with figure 1.

3. Line 216-218: ‘In case of imbalance…’: this sentence is not clear. Is this imbalance shown to happen in isolated islets? And what do you mean by ‘the functional demand’?

4. Line 230-232: ‘We find activation…’: this sentence is not clear, what do you mean with this sentence?

5. Line 248-258 about mitochondria don’t seem relevant for this review, the authors can assume that the readers will know these basics about mitochondria.

6. The reoccurrence of autoimmunity should also be mentioned in figure 2. Figure 2 also mentions inflammation and oxidative stress but they don’t seem to come back in the text.

7. In the revascularization part the authors mention the paper of Kado et al. 2024 (reference 118) and how co-transplantation skeletal myofibroblast is an attractive way to improve vascularization. However, this study was done under the kidney capsule and not into the liver. Do the authors believe co-transplantation is an option when transplanting into the portal vein or is it important to switch to another transplantation site when you want to use these strategies? The authors should also mention the issues associated with co-transplantation of another type of cell.

8. The review is very focused on the liver as transplantation site, however, nowadays there are promising transplantation devices that solve several of the issues associated with the post-transplantation period in the liver. Especially since the authors claim to highlight new avenues, these approaches should be mentioned.

9. The conclusion would be stronger when it describes the most promising new avenues of action.

10. Please check the text carefully for spelling mistakes and typo errors. Also, explain all abbreviations and only do this once, the first time an abbreviation is used. Only abbreviate if you use the word multiple times afterwards.

Comments on the Quality of English Language

The review is sometimes difficult to follow and please check the text carefully for spelling mistakes and typo errors.

Author Response

Dear Reviewer,

You could find below our answers on your comments concerning our manuscript entitled: "Islet transplantation: current limitations and challenges for successful outcomes" by Allan Langlois, Michel Pinget, Laurence Kessler, Karim Bouzakri, which was reviewed as an Review article in Cells: cells-3254458.

All your comments have been addressed by making changes to the manuscript (indicated by line numbers in this letter and highlighted in yellow in the manuscript). We are grateful for your relevant comments that will enhance the level of our review.

Answer to Reviewer 1 comments:

Is there a word missing at the end of the title? Or should it be ‘challenges for success’? The same for the subtitle: the dark side of islet isolation successful.

Thank you very much for your comments. We changed the title by “Islet transplantation: current limitations and challenges for successful outcomes” (l.1). As well, we corrected the subtitle by “Ischemia: the dark side of islet isolation success” (l.185).

Authors describe in the first part how ECM is important and that it takes time to restore it post-transplantation, they also describe ischemia during isolation, but nothing is said about the disruption of the blood vessel network and that this also takes time to be restored post-transplantation. Vascularization is mentioned in figure 1, so you also expect it in the text associated with figure 1.

Thank you for your comments. Firstly, we added this sentence (l.93 to 95): “Another critical factor to consider in protecting islet preparation is the ischemia faced by the pancreas and pancreatic islets following the disruption of the blood vessel network [21,22]”.

Secondly, we completed the section “Ischemia: the dark side of islet isolation success” with informations concerning the importance of the blood vessel network for islet function and survival and consequences of it disruption (l.186-l.191): “Vascularization is essential for the survival and optimal function of pancreatic islets. As a matter of fact, they are highly vascularized, receiving 6-20% of the direct arterial blood flow, which ensures adequate gas and nutrient exchange as well as waste removal. It allows also a rapid delivery of insulin into the bloodstream in response to rising glucose levels [36]. Moreover, ECM from islet microcapillary endothelial cells affects β-cell spreading and enhances insulin secretion [37]”.

Finally, we added informations on the time required to restore revascularization of islets after transplantation in the “Islet revascularization” section (l.464-l.472): “As previously described, transplanted islets are avascular and rapid restoration of blood flow seems vital to preserve functional β-cell mass. However, this process takes several days to a few weeks [121] and the delayed functional formation of micro vascularization deprives islet cells of oxygen and nutrients, causing their apoptosis and/or necrosis [30,37,122]. Indeed, vascular buds form on the periphery of the islets within 14 days of transplantation, but the core of the islet is still poorly vascularized during this period. It takes about two more weeks for this latter to revascularize and oxygen levels and waste disposal to recover. Finally, the transplanted islets only come close to normal levels of endogenous oxygen after one month post-injection [30]”.

Line 216-218: ‘In case of imbalance…’: this sentence is not clear. Is this imbalance shown to happen in isolated islets? And what do you mean by ‘the functional demand’?

Thank you for your comments. This sentence has been clarified and the section completed (l.227 to l.232): “In the case of ER functional perturbations, it leads to what is known as ER stress, characterized by the accumulation of unfolded or misfolded proteins in the ER lumen resulting in cell dysfunction [58]. Moreover, ER stress has been widely shown to be involved in islet dysfunction across the diabetes spectrum, as both ER trafficking and protein folding capacity are integral to β-cell protein quality control and insulin synthesis [59,60]”.

Moreover we mentioned Omori K's work, who demonstrated the induction of ER stress in isolated islets caused by prolonged cold ischemia (l.247 to l.252): “Interestingly, Omori K et al [68] demonstrated by electron micrographs, that prolonged cold ischemia induced severe swelling and disruption of cell and mitochondrial membranes, as well as an expanded ER in isolated β-cells. The authors also found that the process of isolating islets, independently of the cold ischemia of the pancreas, activated the UPR, while BiP protective chaperon was even more overregulated by cold ischemia/pancreas warming. Therefore, this response participates in the death of pancreatic islets by apoptosis and necrosis”.

Line 230-232: ‘We find activation…’: this sentence is not clear, what do you mean with this sentence?

Thank you for your comment. This sentence has been rewritten (l.240 to l.243): “This allows notably the maintenance of the integrity and normal β-cells function and is classically controlled by three canonical ER-resident transmembrane proteins, the activation factor 6 (ATF6), the inositol requiring 1, and the endoplasmic eIF2a kinase PKR/pancreatic (PERK/PEK) [57,58]”.

Line 248-258 about mitochondria don’t seem relevant for this review, the authors can assume that the readers will know these basics about mitochondria.

Thank you for your comment. This part has been deleted. The section entitled “Mitochondrial dysfunction in pancreatic β-cells leads to impaired insulin secretion and apoptosis” begins now l.265.

The reoccurrence of autoimmunity should also be mentioned in figure 2. Figure 2 also mentions inflammation and oxidative stress but they don’t seem to come back in the text.

Thank you for your comment. Figure 2 has been modified.

In the revascularization part the authors mention the paper of Kado et al. 2024 (reference 118) and how co-transplantation skeletal myofibroblast is an attractive way to improve vascularization. However, this study was done under the kidney capsule and not into the liver. Do the authors believe co-transplantation is an option when transplanting into the portal vein or is it important to switch to another transplantation site when you want to use these strategies? The authors should also mention the issues associated with co-transplantation of another type of cell.

Thank you for your comments. This part was added in the text l.505 to l.513: “Given the current knowledge about the beneficial effects of cross-cell communication involving a variety of factors, co-transplantation appears to be a promising approach for improving efficiency in island transplants. Nevertheless, further research is needed on the safety of co-transplantation, as there are still many questions to be clarified regarding the fate and medium- or long-term effect of undifferentiated cells on the patient, on the graft and the function of the organ receiving the cell preparation. In view of these questions that remain pending, we can also question its possible use in the liver due to the risk of hemorrhage related to intraportal injection which may lead to the spread of undifferentiated cells throughout the body”.

The review is very focused on the liver as transplantation site, however, nowadays there are promising transplantation devices that solve several of the issues associated with the post-transplantation period in the liver. Especially since the authors claim to highlight new avenues, these approaches should be mentioned.

Thank you for your suggestion. Informations were included in the “Islet transplantation and immunosuppression” section (l.386 to l.398): “In addition, remarkable progress has been made in recent years on the immuno-isolation of pancreatic islets by encapsulation. Indeed, a number of medical devices have been able to demonstrate their effectiveness such as Beta-O2 device [101,102] which consists in a container with a PTFE semi-permeable membrane inside which islets are embedded in an alginate hydrogel. This device allowed maintaining graft function up to 10 months, but with low levels of circulating C-peptide and with no impact on metabolic control [103]. One drawback to this system is the need to fill it with O2 every day, which can be a constraint for the patient. Then, islet nano-encapsulation could be also a promising option for conferring immunological protection to the islet. Moreover, thanks to an ultra-thin layer of encapsulating material this approach allows easy oxygen and nutrient inflow and insulin outflow [103,104]. Another advantage of this approach would be the possibility to inject these encapsulated islets into the liver and benefit from the first-pass effect. However, further studies on efficacy and safety are still needed”.

The conclusion would be stronger when it describes the most promising new avenues of action.

Thank you for your comment. We modified the conclusion (l.592 to l.605): “Throughout this article, we have aimed to provide an overview of recent findings on the deleterious mechanisms affecting pancreatic islet quality during the isolation process (Figure 1), and to present proposals to address these issues. Additionally, we discussed challenges (Figure 2) and potential solutions for transplanted islets. This allowed us to appreciate the numerous complications that pancreatic islets face during the various stages of transplantation. As a result, there is still a great deal of work to be done to protect the functional mass of islets and reduce the number of pancreases needed for a single patient. Moreover, the standardization of the complete procedure between the different centers remains one of the key issues to be solve in order to optimize the success of the islet transplantation. However, the remarkable scientific advancements in the past few years in understanding these deleterious pathways have enabled the identification of numerous targets for action and the establishment of various strategies (genetic, pharmacological, organoid, encapsulation, stem cells), all of which are equally promising. Ultimately, these advances bring hope that more T1DM patients will be treated in the near future”.

Please check the text carefully for spelling mistakes and typo errors. Also, explain all abbreviations and only do this once, the first time an abbreviation is used. Only abbreviate if you use the word multiple times afterwards.

Thank you very much for your comments. These have been taken into consideration.

Comments on the Quality of English Language

The review is sometimes difficult to follow and please check the text carefully for spelling mistakes and typo errors.

Thank you very much for your comment. We reviewed the overall of the manuscript by an experienced English-speaking colleague to improve it quality.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript will be valuable but it is extremely difficult to read due to poor grammar that starts with the tile. The introduction was the most difficult to read and the grammatical error distracted from the content.

Title— successful is an adjective and must modify a noun

consider

current limitations and challenges for success

current limitations and challenges for successful outcomes

Introduction

I would recommend a re-write as there are over 20 grammatical errors in this section. Incorrect verb tenses, run on sentences, awkward transitions. This makes it very difficult to appreciate content.

Importance of ECM section has less errors

Happy to further read after grammatical editing performed

Comments on the Quality of English Language

Moderate revisions required

Author Response

Answer to Reviewer 2 comments:

Title— successful is an adjective and must modify a noun consider current limitations and challenges for success or current limitations and challenges for successful outcomes

Thank you very much for your comments. Considering your pertinent proposition, we changed the title by “Islet transplantation: current limitation and challenges for successful outcomes”.

Introduction

I would recommend a re-write as there are over 20 grammatical errors in this section. Incorrect verb tenses, run on sentences, awkward transitions. This makes it very difficult to appreciate content.

Thank you very much for your comments. The introduction has been rewritten (l.30 to l.86): “Type 1 diabetes mellitus (T1DM), or insulin-dependent diabetes, is an autoimmune disease characterized by dysfunction of T lymphocytes, responsible for destroying the β-cells of the pancreas. As a result, insulin production becomes insufficient or non-existent, leading to a prolonged hyperglycemia [1], and with the main treatment consists of subcutaneous injections of insulin several times a day. However, this strategy is sometimes insufficient to effectively regulate glycemia and necessitating alternative therapies. One of these alternatives, is pancreatic islet transplantation. This minimally invasive strategy is indicated for unstable T1DM patients suffering from recurrent hypoglycemia, impaired awareness of hypoglycemia and those who do not respond to intensive medical treatment. It is also performed after kidney transplantation in cases of end-stage renal failure [2–4]. Current clinical data show that islet transplantation is a sound therapeutic option for patients with T1DM, stabilizing glucose metabolism, normalizing glycemia, and considerably reducing episodes of severe hypoglycemia. In addition, the complications of diabetes are delayed or attenuated and are associated with an improvement in diabetes-related quality of life [2,3,5,6]. Furthermore, a recent clinical study demonstrated the beneficial effect of islet transplantation on kidney graft survival in T1DM patients [7]. Moreover, islet transplantation has recently been shown to improve cognitive impairment associated with glycemic imbalance [8].

Pancreatic islet transplantation involves several stages. It begins with the removal of a pancreas from a brain-dead donor, which is then rapidly transferred to the cell therapy unit to isolate the islets. A collagenase solution is injected into the pancreas via the Wirsung duct, and digestion is carried out in a Ricordi chamber under mechanical agitation. The islets are then purified using density gradient centrifugation of the digesta. At the end of this procedure, the islet preparation is assessed in terms of purity, viability, sterility, number and, sometime for functionality [9]. Current recommendations for clinical perfusion are at least 3,500 Islet Equivalent Quantity (IEQ)/kg of recipient body weight (with a total of at least 10,000 IEQ/Kg for a complete transplant procedure), viability greater than 80% and purity greater than 50% if possible [10]. Nevertheless, this consensus may be modified in countries where the procedure is now covered by the healthcare system. Nowadays, it is well known that the main objective of islet transplantation is not necessarily to achieve insulin independence. Indeed, the current outcome for optimal graft function are an HbA1c <7.0% without severe hypoglycemia, a >50% reduction in insulin requirements and restoration of clinically significant C-peptide [11,12]. Nonetheless, these goals remain difficult to achieve as islet transplantation encounters numerous obstacles throughout the various stages of transplantation, resulting in a significant loss of viable and functional pancreatic β-cell mass. Indeed, the quantity of functional pancreatic islets continues to decrease throughout the entire process, from the donor stage to the post-transplant period. According to the literature, this can be explained by the induction of cell death mechanisms, by inflammatory reactions and hypoxia induced before and after transplantation [13–17]. This is also due to host immune rejection reactions, the undesirable effects of the immunosuppression used to combat them [18] and by the recurrence of post-transplant autoimmunity [19,20]. As a result, it takes an average of 2 to 3 pancreases to treat one patient, making it difficult to offer islet transplantation to all patients with T1DM who requiring it.

To cope with the excessive number of pancreases required for one patient, it is crucial to protect and preserve the functional mass of pancreatic islets from the earliest stages of transplantation. Therefore, it is important to understand how these deleterious phenomena affect islet number, function and survival of pancreatic β-cells in order to develop graft protection strategies. With that in mind, this manuscript presents an overview of the detrimental mechanisms affecting both the quality of pancreatic islets for transplantation and, the survival and function of engrafted islets. Specifically, we highlight in here the importance of the extracellular matrix (ECM) for optimal β-cell function and survival. We then examined the deleterious effects of ischemia, with focus on endoplasmic reticulum (ER) stress and mitochondrial dysfunction. Furthermore, after transplantation, we addressed the problem of rejection and the role of immunosuppression, Instant Blood-Mediated Inflammatory Reaction (IBMIR), islet revascularization, and the peri-transplant insulin management. We also explore the potential role of cellular plasticity in graft efficacy and have sought to present examples of solutions to address all of these issues. Finally, we hope that this review will give insight into new avenues for action, allowing islet transplantation to be offered to a larger number of patients with T1DM”.

Importance of ECM section has less errors

Happy to further read after grammatical editing performed

Comments on the Quality of English Language

Moderate revisions required

Thank you very much for your comment. We reviewed the overall of the manuscript by an experienced English-speaking colleague to improve it quality.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors sufficiently adjusted the manuscript after the rebuttal.

Comments on the Quality of English Language

Although the authors claim that an English-speaking collegue checked the manuscript, there is a mistake in line 508 'island', while this should be islet (essential for this paper).

Author Response

ANSWER TO THE REVIEWER 1

Dear Reviewer,

Your comment has been addressed by making changes to the manuscript (indicated by line numbers in this letter and highlighted in yellow in the manuscript). We are grateful for your relevant comment that will enhance the level of our review.

Answer to Reviewer 1 comments:

Although the authors claim that an English-speaking collegue checked the manuscript, there is a mistake in line 508 'island', while this should be islet (essential for this paper).

Thank you for your comment. “Island” has been changed by “Islet” l-513.

Reviewer 2 Report

Comments and Suggestions for Authors

This version of the submitted manuscript is easy to read and understand . The complex items that comtribute to beta-cell failure have been presented in an organized fashion. All of the grammar issues identified in the first version have been resolved. I have on minor comments.

Title- Suggested correction has been incorporated

Line 14--Abstract is very well written

Line 43--Current clinical data... sentence needs a reference

Line 58--Glad the authors point out that the goal of islet transplantation is not just insulin independence.

Line 233--change "its" to insulin.

Line 302--Could you please define Imeglimin and its mechanism of action? My quick search says it is a blocker of oxidative phosphorylation and an oral medication used for T2DM.

Line 329-- Change the word "study" to review or document

Line 345--Consider the following change. Glucocorticoid free immunosuppression or Immunosuppression free of glucocorticoid

Line 534--Could you include glycemic targets post islet transplantation (example 80-125 or 80-140 mg/dl) and reference for this population?

Line 537--Could you include any human references to support improved outcomes with intensive exogenous insulin therapy. This paragraph is not as well supported with data as the others.

Line 580--Nice summary and provocative/interesting hypothesis presented

Thank you for this effort

Author Response

Dear Reviewer,

All your comments have been addressed by making changes to the manuscript (indicated by line numbers in this letter and highlighted in yellow in the manuscript). Thank you very much for your encouraging comments and we are grateful for your relevant remarks that will enhance the level of our review.

Answer to Reviewer 2 comments:

Line 43--Current clinical data... sentence needs a reference

Thank you very much for your comment. Three references have been added l.43.

Line 233--change "its" to insulin.

Thank you very much for your comment. ‘Its” has been changed by “insulin” l.233.

Line 302--Could you please define Imeglimin and its mechanism of action? My quick search says it is a blocker of oxidative phosphorylation and an oral medication used for T2DM.

Thank you for your comment. This part has been added l.304 to l.309: “Imeglimin is a tetrahydrotriazine-containing class of oral glucose-lowering agents synthesized from Metformin, which reduces glucose and lipid biosynthesis in the liver by suppressing anabolic processes; and increases glucose uptake in skeletal muscle [81]. It is also known to normalize glucose tolerance and improve insulin sensitivity by protecting mitochondrial function from oxidative stress and favoring lipid oxidation in the livers of mice fed a high-fat, high-sucrose diet [81,82].”

Line 329-- Change the word "study" to review or document

Thank you for your comment. “study” has been changed by “review” l.334.

Line 345--Consider the following change. Glucocorticoid free immunosuppression or Immunosuppression free of glucocorticoid

Thank you for your comment. “an immunosuppression free glucocorticoid” has been changed by “Glucocorticoid free immunosuppression” l.350.

Line 534--Could you include glycemic targets post islet transplantation (example 80-125 or 80-140 mg/dl) and reference for this population?

Thank you for your comment. This part has been added l.542 to l.545: “Moreover, insulin therapy is generally implemented when random glycemic sampling demonstrates on three subsequent occasions within the same week fasting values >140 mg/dl (7.8 mmol/L) and postprandial values >180 mg/dl (10.0 mmol/L), or after recording two consecutive A1c values >6.5% [131]”.

Line 537--Could you include any human references to support improved outcomes with intensive exogenous insulin therapy. This paragraph is not as well supported with data as the others.

Thank you for your comment. We have sited Shapey I M et al’s work l.542 (Ref 133).